The role of dolichol monophosphate in sugar transfer

The specificity of the transfer of monosaccharides from sugar nucleotides to dolichol monophosphate catalyzed by liver microsomes was studied. Besides uridine diphosphate glucose, uridine diphosphate-N-acetylglucosamine and guanosine diphosphate mannose were found to act as donors for the formation of the respective dolichol monophosphate sugars. Uridine diphosphate galactose and uridine diphosphate-N-acetylgalactosamine gave negative results.     

Differential effect of inflammation and dexamethasone on dolichol and dolichol phosphate synthesis

Inflammation and glucocorticoids stimulate hepatic glycoprotein synthesis, resulting in an increased secretion of serum glycoproteins. We now present evidence that the synthesis of dolichol and dolichol phosphate from mevalonate is increased in hepatocytes from inflamed rats. Also, in inflamed rats, the levels of dolichol and dolichol phosphate are increased in liver homogenates and microsomes. Dexamethasone treatment of the cells, however, does not increase the synthesis of dolichol and dolichol phosphate from mevalonate. The results suggest that the inflammation-induced dolichol-linked saccharide and glycoprotein synthesis is possibly mediated through an increase in the level of dolichol and dolichol phosphate in the liver. Since dexamethasone treatment does not increase the synthesis of dolichol and dolichol phosphate, its action on glycoprotein synthesis appears to be different and to affect the induction of enzymes in mannosyl phosphoryl dolichol- and dolichol-linked oligosaccharide synthesis.     

Comparative studies on mannosylphosphoryl dolichol and glucosylphosphoryl dolichol synthases

Factors affecting the synthesis of mannosylphosphoryl dolichol and glucosylphosphoryl dolichol hen oviduct microsomes were compared in order to gain insight into the properties of their respective synthases. A stabilized form of mannosylphosphoryl dolichol synthase, but not glucosylphosphoryl dolichol synthase, was released from microsomes by freezing the membranes after exposure to the detergent CHAPSO. The activation energy for mannosylphosphoryl dolichol synthesis in membranes was 9.4 glucosylphosphoryl dolichol synthesis in membranes had a similar activation energy, 8.1 kcal/mol, but below 18 degrees C the value was 16.7 kcal/mol. Tryptic digestion of sealed microsomes preferentially inactivated mannosylphosphoryl dolichol synthase; however, both synthases were equally inactivated in detergent-permeabilized microsomes. Periodate-oxidized UDP-Glc was used to probe the topological orientation of glucosylphosphoryl dolichol synthase in rat liver microsomes. Sealed microsomes treated with oxidized UDP-Glc were inactive in synthesis of glucosylphosphoryl dolichol. However, when these treated microsomes were permeabilized, glucosylphosphoryl dolichol synthase activity was readily detected. From these studies we conclude that although mannosyl- and glucosylphosphoryl dolichol synthases catalyze chemically similar reactions in the endoplasmic reticulum, they differ in several respects. These differences were interpreted in terms of a topological model in which the active sites of the two enzymes reside on opposite faces of the endoplasmic reticulum, with that of the glucosyl lipid synthase facing the lumen and that of the mannosyl lipid synthase facing the cytosol.     

Stimulation by dolichol phosphate-mannose and phospholipids of the biosynthesis of N-acetylglucosaminylpyrophosphoryl dolichol

Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean, E. L. (1982) J. Biol. Chem. 257, 7952-7954). Further studies on this phenomenon are described, using microsomes from the retina of the embryonic chick as the major source of enzyme. Neither dolichol-P-glucose nor retinyl-P-mannose showed this stimulatory activity. Phosphatidylglycerol also stimulated this same process and was most active among a variety of phospholipids which were tested, in accord with previous reports. The presence of GDP-2-deoxy-2-fluoro-D-mannose or GTP had no effect on the reaction. The apparent activation constant for dolichol-P-mannose was 2.2 microM, and for phosphatidylglycerol, 401 microM. The major product (90% or greater) obtained under basal and stimulatory conditions was GlcNAc-P-P-dolichol and the site of the stimulatory effect was the glucosaminyltransferase catalyzing the formation of this compound. The effects of stimulation on the kinetic properties were similar for both activators: increases in the Vmax of the reactions of 7-10-fold; increases in apparent Km for UDP-GlcNAc of 5-7-fold; a 3-fold decrease in apparent Km for dolichol-phosphate. When present together, a mutual inhibition of stimulation was observed compared to the additive effect from dol-P-mannose or phosphatidylglycerol alone. Although a substrate for the reaction, dolichol phosphate repressed the stimulation by dolichol-P-mannose but not that by phosphatidylglycerol. Dol-P-glucose, while not an activator of the reaction, acted as a negative modifier of the stimulation by dol-P-mannose by acting as a competitive inhibitor of the stimulation. The stimulatory phenomenon was observed in microsomes prepared from a variety of tissues from the embryonic chick and from postnatal tissue after partial delipidation. The addition of pyrophosphatase inhibitors did not bring about stimulation of GlcNAc-lipid synthesis, but did enhance the effect. These studies extend the previous observations of the participation of dolichol-P-mannose and phosphatidylglycerol as allosteric activators of GlcNAc-lipid synthesis and indicate additional aspects of metabolic regulation of the dolichol pathway.     

The high-performance liquid-chromatographic analysis of ficaprenol and dolichol.

A high-performance liquid-chromatographic method was devised which is capable of resolving the p-nitrobenzoyl derivatives of polyprenols containing 35-110 or more carbon atoms. This procedure was used for the determination of ficaprenol and pig liver dolichol composition and can be applied to mixtures of polyprenols as an analytical or preparative technique.     

The esterification of dolichol by rat liver microsomes.

The incubation of 1-[3H)dolichols with cell-free preparations from various rat tissues resulted in the formation of a labeled material which possessed the characteristics of synthetic dolichol palmitate. Rat liver microsomes were found to be a good source of the acyltransferase activity, and the properties of the reaction were investigated using microsomal preparations. The reaction did not require ATP, CoA, or Mg2+ and was stimulated by the addition of phosphatidylcholine. The esterification of dolichol appears to be similar to the esterification of retinol. The fact that the esterification of dolichol is not depressed even in the presence of a several-fold excess of retinol is evidence that the two reactions are catalyzed by different enzymes.     

The alpha-saturation and terminal events in dolichol biosynthesis

Incubations of 10,000 X g supernatant from rat liver with [3H]mevalonate were performed and the labeling of polyprenols was studied. It was demonstrated that factors like pH, substrate concentration, and presence of detergent not only greatly influence the total incorporation but also the relative distribution of radioactivity among the isoprenologues. The synthesis was shown to be extremely sensitive to Triton X-100. Substrate concentrations of 1 and 100 microM mostly gave polyprenols with 18 and 20 isoprenes, respectively. At a given substrate concentration, pH 6.5 resulted in shorter polyprenols than did pH 7.5. Ozonolytic fragmentation demonstrated that in the initial phase of incubation, polyprenols are elongated by 1 isoprene residue and saturated to give dolichols. No substantial dephosphorylation of polyprenyl phosphates to the free alcohol occurred. The production of dolichol in vitro was shown to utilize NADH for the saturation event. This seemed to occur concomitantly with the synthesis. alpha-Saturation of polyprenyl-P could not be achieved with the procedures employed. It is proposed that the synthesis of dolichol and dolichyl-P do not share the same terminal steps; saturation and terminal isoprene condensation occur in cooperation; and substrate concentration and pH influence the terminal enzyme(s) and the nature of the final product in the polyprenol biosynthesis.     

The role of calmodulin in the regulation of dolichol kinase

A calcium ion-requiring CTP-dependent kinase that phosphorylates dolichol was found in particulate enzyme preparations from the protozoa Tetrahymena pyriformis. This enzyme and an analogous enzyme present in rat brain microsomes were both shown to be inactivated following washing with EGTA-containing buffers. The activity could be restored by the addition of calcium and the calcium-binding protein calmodulin. In addition, both enzymes were strongly inhibited by trifluoperazine, chlorpromazine, and antiserum against brain calmodulin. These results are evidence that the dolichol kinase from these two sources is regulated by a system involving calmodulin. Dolichol kinase is the enzyme that is believed to be important in the maintenance of the cellular levels of dolichyl phosphate, the factor which is likely to exert the most control over the rate of glycoprotein biosynthesis. On the other hand, microsomal preparations from rat liver which were shown to contain a dolichol kinase that does not require Ca2+ for activity showed no inactivation by EGTA treatment, trifluoperazine, chlorpromazine, or preincubation with antiserum against calmodulin. These findings indicate that the liver enzyme and thus the level of dolichol phosphate is controlled by a different mechanism than that of brain and T. pyriformis.     

The half-lives of dolichol and dolichyl phosphate in rat liver

Rat liver dolichol and dolichyl-P were labeled by injection of [³H]mevalonate into the portal vein and their rates of synthesis and breakdown determined. In the initial phase the radioactivity appeared in -unsaturated polyprenols. Subsequent saturation required 90 min. The half-lives of dolichols in microsomes were between 80 and 118 h, and shorter dolichols had shorter values of T_1/2. The half-lives of dolichols in lysosomes were between 115 and 137 h, while microsomal dolichyl-P exhibited a T_1/2 of 32 h. Injected dolichol was recovered in the lysomes of hepatocytes and exhibited a rate of breakdown which was slower than that of the endogenous compound. These results indicate differences in the catabolism of dolichol at different subcellular locations, as well as differences between the catabolism of dolichol and dolichyl-P.     

Esterification of dolichol in rat liver

In the various subcellular fractions of rat liver 45-75% of the total dolichol was esterified with a fatty acid. The esterification reaction was localized exclusively in the microsomes, and the transferase activity is 3-fold higher in the cation-insensitive smooth microsomes than in other microsomal subfractions. Although fatty acyl-CoAs tested served as substrates, palmitoyl-CoA was the most rapidly utilized. None of the phosphatidylcholine or phosphatidylethanolamine species tested could be utilized to esterify dolichol with a fatty acid, indicating the absence of transacylation. alpha-Saturated dolichols were esterified at a higher rate than their alpha-unsaturated counterparts. Albumin and low concentrations of Triton X-100 activated the esterification reaction, which was not dependent on mono- or divalent cations, ATP, or CoA. The sensitivity of the transferase activity to trypsin indicates localization of the enzyme(s) involved on the outer surface of microsomes (i.e. the cytoplasmic surface of the endoplasmic reticulum), as is also the case for enzymes of dolichol biosynthesis. Transferase activity was detected in all tissues examined but at a much lower level than in liver and testis. The patterns of fatty acids in dolichol esters of different organelles exhibited some specificity. Labeling in vivo indicated that esterification of dolichol may play a role in targeting this lipid from the endoplasmic reticulum to lysosomes.     

The conformation of apamin

Energy minimization techniques are used as a tool to distinguish between different proposed models for the structure of the bee venom polypeptide apamin. The influence of electrostatic interactions on the resultant energies is noted. The model of Hider and Ragnarsson [(1980) FEBS Lett. 111, 189-193] is found to be of consistently low energy.     

Biosynthesis of dolichol by rat liver peroxisomes

The ability of peroxisomes and microsomes to synthesize dolichol from [3H]mevalonate, [3H]isopentenyl-P2 or [3H]farnesyl-P2 in vitro was investigated. It was found that isoprenoid biosynthesis also occurs in peroxisomes and that this process demonstrates properties differing from those of isoprenoid biosynthesis by microsomes. The pH optimum in peroxisomes was 8.0 and, in contrast to microsomes, the peroxisomal biosynthesis was largely insensitive to detergents. After treatment with proteolytic enzymes, microsomes lost their capacity to incorporate [3H]mevalonate into dolichol, whereas proteolysis of intact peroxisomes did not influence their corresponding rate of incorporation. The soluble content of peroxisomes was separated from the membranes and found to demonstrate half of the biosynthetic capacity of the intact organelle. Fasting and cholestyramine treatment decreased only the microsomal incorporation of [3H]mevalonate into dolichol, while treatment with clofibrate, di-2-ethylhexyl phthalate or phenobarbital increased microsomal, but decreased peroxisomal labeling. After injection of [3H]mevalonate into the portal vein of rats, high initial labeling of dolichol was recovered both in isolated microsomes and peroxisomes, whereas when [3H]glycerol was administered, peroxisomal phospholipids became labeled later than the corresponding microsomal constituents. These results support the conclusion that dolichol is synthesized both in peroxisomes and the endoplasmic reticulum, but that the biosynthetic processes at these two locations have different properties.     

Effects of dolichol on membrane permeability

Small vesicles containing the tetra-anionic fluorescent probe calcein were prepared by sonication of mixtures of plant phosphatidylethanolamine, plant phosphatidylcholine, and dolichol. Following chromatography, the isolated vesicles were found to retain entrapped calcein over the temperature range of 15 to 40 degrees C. Utilizing an assay measuring the fluorescence quenching of entrapped calcein by cobalt ions, the presence of dolichol in the membranes was found to promote the permeability of the phospholipid bilayers to the divalent cation. The permeability was shown to be dependent on temperature with an increase in rate of 17-fold between 15 and 35 degrees C although the plant phospholipids used in these experiments have no known phase transition within this temperature range. The incorporated dolichol was distributed uniformly throughout the vesicle population. Similar vesicles prepared from phosphatidylethanolamine and phosphatidylcholine without added dolichol, from phosphatidylcholine alone, or with phosphatidylcholine and dolichol were far less permeable to the divalent cation under the same assay conditions. These results demonstrate that dolichols have significant effects on the permeability properties of phospholipid bilayers that contain phosphatidylethanolamine.     

Hypoglycosylation due to dolichol metabolism defects

Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C- and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for N-glycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.